Green fluorescent protein (GFP) derived from a jellyfish, Aequorea victoria, has many purposes in biological research. Recently, various GFP mutants have been developed using random mutagenesis and semi-rational mutagenesis, wherein a color is changed, a folding property is improved, luminance is enhanced, or pH sensitivity is modified. Fluorescent proteins such as GFP can be fused with other proteins by recombinant DNA techniques to visualize the expression and translocation of the fusion proteins.
One of the most commonly used variants of GFP is yellow fluorescent protein (YFP). Among Aequorea-derived GFP mutants, YFP exhibits the fluorescence with the longest wavelength. The molar extinction coefficient (ε) and fluorescence quantum yield (Φ) of most YFPs are 60,000 to 100,000 M−1 cm−1 and 0.6 to 0.8, respectively (Tsien, R. Y. (1998). Ann. Rev. Biochem. 67, 509-544). These values are comparable to those of fluorescent compounds such as fluorescein and rhodamine. Cyan fluorescent protein (CFP) is another example of a GFP mutant. ECFP (enhanced cyan fluorescent protein) is such a CFP which has been known. Furthermore, red fluorescent protein (RFP) has been isolated from sea anemone (Discoma sp.), and among such red fluorescent proteins, DsRed has been known. Thus, four types of fluorescent proteins including green, yellow, cyan and red fluorescent proteins, have been developed one after another, and their spectrum range has been significantly extended.
In addition, using a fluorescent protein whose color is changed by light irradiation, it becomes possible to optically mark specific cells or organs. For such light irradiation-dependent marking of cells, tissues and the like, photoactivatable green fluorescent protein (PA-GFP) (Patterson G H and Lippincott-Schwartz J, Science 297, 1873-1877 (2002)) and Kaede (Ando R et al, Proc. Natl. Acad. Sci. USA 99, 12651-12656 (2002)) are used. However, since PA-GFP is characterized in that fluorescence appears from a nonfluorescent state, finding the position of a sample before irradiation with stimulating light is problematic. On the other hand, Kaede changes its color from green to red as a result of irradiation with stimulating light. However, Kaede requires excitation lights that depend on both colors, and thus the operation thereby becomes complicated. Moreover, since Kaede forms a tetramer, it is not suitable to observe dynamics of Kaede fused with any given protein.